During the past year, this laboratory continued studies on the mechanism of HIV entry, and on the development of novel protective and treatment strategies based on the molecules involved in entry. 1) Molecular mechanisms of HIV Env-mediated entry/fusion. a) Subunit interactions within the oligomeric structure of HIV Env. We found that Envs containing different functional defects in gp120 or gp41 can complement one another within the HIV oligomer. The results support a model in which cooperative subunit interactions within the Env oligomer result in concerted conformational changes upon receptor binding, resulting in activation for fusion. Future work will focus on the implications of these findings for Env function and virus neutralization. b) Mechanisms of antibody resistance of some primary HIV-1 strains. We have developed assays to assess whether such isolates can be rendered sensitive by pretreating Env with soluble CD4. Our studies are designed to distinguish whether the antibody resistance is due to absence vs. masking of the corresponding epitopes. The results will have important implication for the development of anti-HIV vaccines based on neutralizing antibody. 2) Novel anti-HIV agents based on HIV Env/receptor interactions. a) sCD4-17b, a potent HIV-1 neutralizing agent. sCD4-17b is aa recombinant chimeric protein containing soluble CD4 attached via a long flexible polypeptide linker to a single chain antibody that binds to a CD4-induced epitope of gp120 involved in binding to coreceptor. The protein neutralizes diverse primary HIV-1 isolates, with potency considerably higher than the best neutralizing monoclonal antibodies. Future studies will focus on producing and purifying large amounts of the protein for extensive in vitro analysis, and assessment of protective efficacy in animal models.We have also begun a collaborative effort to engineer Lactobacillus, the natural microflora of the healthy human vaginal tract, to produce the chimeric protein. The goal is to colonize the vaginal tract with the engineered lactobacillus to provide durable protection against sexual transmission of HIV. b) 3b3-PE38, an anti-HIV immunotoxin. In vitro studies indicate that 3b3-PE38 is significantly more potent in vitro than a previous agent CD4- PE40. The immunotoxin efficiently kills HIV-infected cells, and inhibits spreading infection of diverse primary isolates in both primary T cells and macrophages. In vivo studies with rhesus macaques indicate minimal hepatoxicity compared to CD4-PE40, in uninfected and SHIV- infected animals. Future work is focused on developing 3b3-PE38 for Phase I clinical trials in individuals whose virus loads have been effectively suppressed by HAART.